The general objective of this project is to elucidate the molecular mechanisms of Tat transactivation during transcription initiation. Localization of discrete Tat functional domains active in transcription initiation will be determined by site-directed mutagenesis and evaluation of Tat mutants on HIV transcription at different stages of pre-initiation and initiation and by construction and evaluation of GAL4/Tat fusion proteins for their effect on HIV transcription in vitro and in vivo. Dissection of the mechanism(s) by which Tat enhances pre-initiation complex formation during HIV transcription will be accomplished by determination of association and disassociation rates of pre-initiation complex assembly using gel shift and footprinting assays, by determination of Tat-protein interactions using co-immunoprecipitation assays, co-sedimentation analysis, protein filter binding, Tat affinity chromatography and by determination of Tat effects on functional activities of pre-initiation complex components. Determination of Tat-mediated effects on the synthetic step of transcription initiation will be afforded by quantitating the abundance of the first dinucleotide synthesized upon transcription initiation from productive pre-initiation complexes.